Many approaches are used in the preparation of vaccines. For viral diseases, these methods include the use of inactivated viruses, live viruses, live recombinant viruses, viral proteins, viral subunit proteins and the like.
However, many types of vaccines have limited success in part because of the extensive genetic variation from one strain or isolate of an organism to another. Additionally, human or animal antigens involved in pathogenesis, i.e. IgE for allergic reactions, tumor antigens, etc., do not produce beneficial immune responses in the same animal species from which they are derived, and indeed may be treated as self-proteins, and not as antigens.
However, it is desirable to use small peptide sequences or epitopes (i.e., antigenic determinants) representing the biological function of a given antigen as immunogens even in homologous animal species. In theory, these peptides can be used to elicit an immune response when injected into animals. In practice, however, these peptides are often not good immunogens because they are usually small and do not present themselves favorably to the immune system.
An example of such a poorly immunogenic peptide is the second conserved domain of the external envelope glycoprotein (gp120) of the human immunodeficiency virus (HIV). It has been found to be important for HIV infectivity and antibody neutralization. Science, 239:1021-1023, Feb. 26, 1988. Antiserum directed against this region (amino acids 254-274) can neutralize three different isolates of HIV in vitro, without affecting the binding of the virus to T-cell receptor or CD4-positive cells. The conserved domain is believed to be critical in a post binding event during virus penetration and could be a potential target for antibody neutralization. However, it has been observed that this domain is relatively "immunosilent" when it is presented to the immune system in the context of natural HIV infection. This kind of immunosilence suggests that the epitope may be masked, for example, by protein configuration.
Further details of HIV antigens can be found in Rusche, et at., "Antibodies that Inhibit Fusion of Human Immunodeficiency Virus-infected Cells Bind a 24 Amino Acid Sequence of the Viral Envelope, gp120," Proc. Natl. Acad. Sci. USA, 85:3198-3202, May 1988; Bolognesi, "Progress in Vaccine Development Against SIV and HIV," Journal of Acquired Immune Deficiency Syndromes, 3:390-394, Raven Press, Ltd., New York, 1990; Wain-Hobson, "Nucleotide Sequence of the AIDS Virus, LAV," Cell, 40:9-17, January 1985; and Ho, et al, "Second Conserved Domain of gp120 Is Important for HIV Infectivity and Antibody Neutralization," Science, 239:1021-1023, Feb. 26, 1988.
One way to enhance the immunogenicity of epitope peptides is to increase their size, and therefore their recognizability, by linking them in some manner to fusion proteins, macromolecules, or adjuvants.
Colbere-Garapin et at. have reported the preparation of insertion mutants of type poliovirus (Sabin strain), in which additional amino acid sequences (tri- or hexapeptides) are inserted within the neutralization site 1 of the capsid protein VP1. Colbere-Garapin, et al., "Addition of a Foreign Oligopeptide to the Major Capsid Protein of Poliovirus," Proc. Natl. Acad. Sci. USA, 85:8668-8672, November 1989.
Evans, et al., have reported the construction and the characterization of a poliovirus antigenic chimera protein containing an epitope from the transmembrane glycoprotein (gp41) of human immunodeficiency virus type 1 (HIV-1). Rabbit antisera raised by subcutaneous injection of live polio/HIV chimera was shown to be broadly neutralizing against several American and African HIV-1 isolates. Evans et al., "An Engineered Poliovirus Chimaera Elicits Broadly Reactive HIV-1 Neutralizing Antibodies," Nature, 339:385-388, Jun. 1, 1989. However, the use of live viral vectors such as polio virus presents problems for practical use, in that serum antibodies may arrest replication of recombinant virus in the body. In addition, live viral vectors have rigid constraints on the size of an insert and provide a limited number of insertion sites.
Wu, et al., have reported the in-frame insertion of synthetic oligonucleotides corresponding to amino acid residues of S (122-137) and pre-S.sub.2 (120-145), which are hepatitis B virus surface antigens, into the hypervariable region of a cloned Salmonella flagellin gene. Animals immunized with the live recombinant bacteria developed antibodies specific to the hepatitis B virus epitopes as detected by ELISA. Wu, et al., "Expression of Immunogenic Epitopes of Hepatitis B Surface Antigen with Hybrid Flagellin Proteins by a Vaccine Strain of Salmonella," Proc. Natl. Acad. Sci. USA, 86:4726-4730, June 1989. The immune response was weak, short-lived, and declined rapidly after immunization.
Cohen reported in Science, 262 980 (Nov. 12, 1993), the comments of speakers at the annual AIDS vaccine conference to the effect that although effective immunization toward HIV that was grown in laboratory cell lines may be achieved, immunization toward freshly harvested HIV was not so effected. From this report, even though Barbara Potts from New York's United Biomedical Inc. reported some success, one may conclude that development of strain specific vaccines or vaccine cocktails designed to immunize against a range of HIV antigens may be necessary for effective immunization.
Michel, et al., have reported the insertion of various HIV envelope fragments into the hepatitis B virus surface antigen. Immunization of rabbits with these fusion proteins produced neutralizing antibodies. Michel, et al., "Induction of Anti-Human Immunodeficiency Virus (HIV) Neutralizing Antibodies in Rabbits Immunized with Recombinant HIV-Hepatitis B Surface Antigen Particles," Proc. Natl. Acad. Sci. USA, 5:7957-7961, 1988.
Similarly, it has been found that histamine release from rat mast cells, both in vitro and in vivo, can be inhibited by immunization with a human peptide-protein conjugate. Stanworth, et al., "Allergy Treatment with a Peptide Vaccine," The Lancet, 36:1279-1281, Oct. 7, 1990. See also, Stanworth, et al., "The Role of High and Low Affinity IgE Receptors in Cell Signalling Processes," Molecular Immunology, 27(12):1291-1296, 1990.
Monoclonal antibodies (mAbs) have also been widely used for passive immunization and for diagnostic purposes to detect infections or pathologic conditions like the presence of tumor antigens. However, production of a monoclonal antibody of desired specificity is laborious and unpredictable. Several steps of blind screening and assays involving a series of overlapping peptides are needed to determine specificity of an mAb. Sometimes the functional epitope may not be in the immunodominant position for the antigen to produce an mAb against that region.
Thus, there is a need for vaccines, diagnostic agents, and therapeutic agents that can take advantage of the potential immunogenicity of antigenic determinants or epitopes according to the invention.